The branching tree of intrahepatic bile ducts is lined by a monolayer of epithelial cells, known as cholangiocytes. During development hepatocytes and cholangiocytes arise from bipotential hepatoblast precursors. Certain hepatoblasts, which are destined to become cholangiocytes, form a monolayered ring surrounding the portal mesenchyme. This monolayer of biliary precursors becomes a double layered structure. Lumens then develop between the two layers. Eventually these structures remodel into a branching tree of intrahepatic bile ducts. We have developed a three dimensional culture system that recapitulates important events in early development. This enables us to study the these events with much greater mechanistic detail than would be possible in vivo. We use a stable mouse cell line termed Hepatic Progenitor cells Proliferating on Laminin, (HPPL), which is capable of differentiating into hepatocytes and cholangiocytes. We grow these as a monolayer on top of a layer of extracellular matrix. We then overlay the monolayer with a second layer of extracellular matrix. After overlay some of the HPPL cells move up out of the original monolayer and crawl on top of the original monolayer. These migrating cells eventually form a second monolayer on top of the first. Lumens then form between the two monolayers. This process closely resembles the early events in bile duct formation in vivo. However, migration of the cells out of the original monolayer requires phosphatidyl inositol 3- kinase (PI3K), the enzyme that generates phosphatidyl inositol 3,4,5-trisphosphate (PIP3), as well as the kinase Akt, which is downstream of PIP3. We will study how PI3K and PIP3 are involved in migration of cells out of the monolayer. We will examine the role of Akt, its effector GSK-3 and downstream processes during tubulogenesis. We will study how after migration the cells redifferentiate to form a second layer of polarized epithelial cells. We will test the role of the lipids PIP3 and , phosphatidyl inositol 4,5-bisphosphate in this process. Formation of bile ducts is essential to life. Many diseases involve developmental malformation of bile ducts, while other diseases involve damage and/or regeneration of bile ducts. The proposed work will greatly increase our understanding of early bile duct development and lead to improved health for patients with these diseases. PUBLIC HEALTH RELEVANCE: Formation of bile ducts is essential to life. Many diseases involve developmental malformation of bile ducts, while other diseases involve damage and/or regeneration of bile ducts. The proposed work will greatly increase our understanding of early bile duct development and lead to improved health for patients with these diseases.